The present invention relates to a chemical reaction apparatus for synthesizing an array of compounds. In particular, the present invention relates to a microfluidic chemical reaction apparatus for a combinatorial synthesis.
Methods for synthesizing polymers (e.g., oligonucleotides) on a solid support have been developed for producing large arrays of polymer sequences on solid substrates. These large xe2x80x9carraysxe2x80x9d of polymer sequences have wide ranging applications and are of substantial importance to many industries including, but not limited to, the pharmaceutical, biotechnology and medical industries. For example, the arrays can be used in screening large numbers of molecules for biological activity, e.g., receptor binding capability. And arrays of oligonucleotide probes can be used to identify mutations in known sequences, as well as in methods for de novo sequencing of target nucleic acids. In addition, PNA (peptide nucleic acids) arrays can be used to screen molecules which are useful in antisense (mRNA) gene regulation, or molecules which bind to specific sequences of double-stranded DNA.
Oligonucleotide arrays with up to tens of thousands of samples on an area of a few square centimeters have been synthesized and proven to be extraordinarily useful in various applications including gene expression studies. Over the past several years, a new set of technologies have emerged for making arrays of synthetic surface-bound polymers.
One of the most successful methods of synthesizing high-density patterns on surfaces is photolithography as discussed in U.S. Pat. No. 5,143,854, issued to Pirrung et al., and PCT Application No. 92/10092. In this method, light is directed to selected regions of a substrate to remove protecting groups from the selected regions of the substrate. Thereafter, selected molecules are coupled to the substrate, followed by additional irradiation and coupling steps. By activating selected regions of the substrate and coupling selected monomers in precise order, one can synthesize an array of molecules having any number of different sequences, where each different sequence is in a distinct, known location on the surface of the substrate. This method requires specialized reagents (e.g., photoremovable protecting groups), which presently have significantly lower coupling yield than conventional reagents. Moreover, in general, to make an array of N-mers requires 4N cycles of deprotection and coupling, one for each of the 4 bases, times N base positions. This photolithographic method also typically requires 4N masks, thereby adding a considerable expense to the procedure.
Another method for producing polymer arrays is an ink-jet technique which uses the print heads of commercial piezoelectric ink-jet printers to deliver reagents to individual spots on the array. While this technique uses relatively inexpensive conventional chemical reagents with typically high coupling yield, it can deliver one, and only one, drop of reagents at a time, unless multiple jets are used simultaneously. Moreover, the solid support must be patterned to achieve small feature sizes. Furthermore, two drops of liquid applied too closely together on a surface tend to spread into each other and mix, thereby limiting the array density achievable with the ink-jet method.
There are other methods including robotic deposition of reagents in an array of fluid-containing wells and the use of fluidics to deposit reagents on a surface. See for example, U.S. Pat. No. 6,001,311, issued to Brennan et al. and U.S. Pat. No. 6,121,048, issued to Zaffaroni et al. However, each method has its own limitations such as limited array density, increased production cost per array, and/or serial (i.e., non-parallel) synthesis.
Therefore, there is a need for a chemical reaction apparatus and a method for preparing array of compounds with high throughput, high product quality, enhanced miniaturization and lower costs.
The present invention provides a chemical reaction apparatus capable of synthesizing an array of compounds.. ,Preferably, the chemical reaction apparatus is a microfluidic system. In one aspect, the present invention provides a chemical reaction apparatus having on/off valves (i.e., switching valves) made out of one or more elastomers. The chemical reaction apparatuses of the present invention are ideally suited for controlling, channeling, and/or directing fluid movement to allow combinatorial synthesis of an array of compounds on a solid support.
In one embodiment, the chemical reaction apparatus of the present invention comprises a solid support base and a first elastomer layer interconnected, i.e., attached thereto. In addition, there is a first plurality of flow channels in between the interface of the solid support base and the first elastic member. In this manner, a solvent, a solution or a reagent can be introduced through the first flow channels to either remove the reagent(s) or to introduce the reagent(s). Moreover, at least a portion of the solid support base within the first flow channels comprises a functional group. This functional group allows one to attach a first group of compound(s) to the solid support base for a subsequent combinatorial synthesis of an array of compounds.
The first elastomer layer may be removably attached to the solid support base. Thus, after a first set of compounds are introduced into a selected plurality of flow channels to attach at least a portion of the compounds on to the solid support base by bonding to the functional groups, the unattached (i.e., non-reacted or non-bonded) compounds are removed from the first flow channels, e.g., by rinsing the plurality of flow channels with a solvent. The elastic member is then removed from the solid support base and reattached to the solid support base such that the first flow channels are oriented at an angle to the previous first flow channel direction, preferably at an angle substantially perpendicular to the previous first flow channel direction. A second set of compounds are then introduced through a selected plurality of flow channels, and again the unattached compounds are removed from the flow channels. The removal and reattachment of elastomer layer and introduction of a set of compounds are repeated until a desired array of compounds are synthesized on a selected portion of the solid support base.
Alternatively, the chemical reaction apparatus of the present invention can have a second plurality of flow channels, which intersect the first plurality of flow channels, preferably at a right angle, i.e., the first plurality of flow channels are perpendicular to the second plurality of flow channels. By selectively closing off either the first or the second plurality of flow channels (or portions thereof), and introducing a set of compounds to the open flow channels, one can prepare an array of compounds on selected portions of the solid support base. Preferably, the array of compounds are synthesized on or near the intersections of the first plurality of flow channels and the second plurality of flow channels. This method provides a variety of advantages including, among others, (1) synthesis of polymers without the need for removal and precise realigning of the first elastic member, and (2) elimination of exposure of growing polymers to air or other contaminants.
In another embodiment of the present invention, one or more additional elastomer layers are attached on top of the first elastomer layer such that recesses (i.e., a plurality of pressure channels) are formed in between each of the various elastomer layers. These pressure channels act as valves by selectively closing a particular flow channel(s) when the appropriate pressure channels are pressurized. In one particular embodiment of the present invention, as illustrated in FIG. 12, at least a portion of the pressure channels (i.e., adjacent pressure channels) are situated (i.e., aligned) in between the first flow channels. In this manner, when pressure is applied to the adjacent pressure channels, at least a portion of the second plurality of flow channels are closed while the plurality of first flow channels remain open. This allows selective addition of reagents into the first plurality of flow channels.
Furthermore, the chemical reaction apparatus of the present invention can also have a plurality of pressure channels (i.e., top-aligned pressure channels) which are situated on a portion, preferably on top, of the first flow channels. In this manner, when pressure is applied to the top-aligned pressure channels, at least a portion of the first plurality of flow channel are closed while leaving the second plurality of flow channels open. Typically, the portions which are open and closed are dependent upon the diameter of the overlying pressure channel.
One of the major advantages of having both top-aligned and adjacent-aligned pressure channels is that by appropriately designing the pressure channels only one additional elastomer layer (i.e., the second elastomer layer) is needed to control all the flow channels.
The elastomer layers can be interconnected by any of the variety of known methods, including bonding together two separate layers with each layer being separately manufactured, e.g., by casting from a micro-machined mold. In one particular embodiment of the present invention,.each layer of the elastic member is separately cured before one elastic member is positioned on top of the other elastic member. The two elastic members are then bonded together, e.g., by using an adhesive or when each elastic member comprises an excess of different component of a two-component curing material, the two elastic member layer can be cured to provide a single elastic piece. In the latter method, each elastic member preferably has an excess of one of the two components or a deficiency of one of the components, such that reactive molecules remain at the interface between the layers. One of the elastic members is assembled on top of the other elastic member and heated or exposed to UV light, for example, to bond one elastic member to the other. The two layers bond irreversibly such that the strength of the interface substantially equals the strength of the bulk elastomer. This creates a single three-dimensional patterned structure from the original two elastic members. Additional elastic layers can be added by simply repeating the process, where new elastic layers, each having an opposite xe2x80x9cpolarityxe2x80x9d are cured, and thereby bonded together.
A further advantage of either above embodiment of the present invention is that due to its integral nature, (i.e., all the elastic members are composed of the same material) the inter layer adhesion failures and thermal stress problems are reduced or completely avoided.
In various aspects of the invention, a plurality of pressure channels pass through the elastic member structure with the pressure channels extending across and above the flow channels. In this aspect of the invention, a thin layer of elastomer separates the pressure channels and the flow channels. As explained in detail below, downward movement of this thin elastomer layer (e.g., due to the pressure channels being pressurized or the thin elastomer layer being otherwise actuated) will cut off flow passing through the flow channels.
In other optional preferred aspects, magnetic or conductive materials can be added to make layers of the elastomer magnetic or electrically conducting, thus enabling the downward movement of the thin elastomer layer using an applied voltage or a magnetic field.
The present invention also provides methods for preparing arrays of polymer sequences wherein each array includes a plurality of different, positionally distinct polymer sequences having known monomer sequences. In one embodiment, the solid support base includes a functional group within at least a portion of the first plurality of flow channels for attaching compounds thereto. While the solid support base need not naturally contain this functional group, it should be appreciated that the solid support base should readily be able to be derivatized to provide appropriate functional groups on the surface for derivatization. Polymer sequences are then synthesized on the surface of the solid support base by selectively exposing a plurality of selected regions on the surface with a monomer, preferably as a solution in a solvent, to couple monomers to the surface in the selected regions. By pressurizing certain pressure channels, one can prevent certain portions of the solid support base from being contacted with a monomer, i.e., one can selectively close off desired flow channels by pressurizing the appropriate pressure channels. The closing of portions of flow channels and contacting the remaining flow channels can be repeated until a plurality of polymer arrays are formed on the surface of the solid support base. Each polymer array includes a plurality of different polymer sequences coupled to the surface of the solid support base in a different known location.
In a further embodiment, the present invention provides a method of synthesizing polymers on solid support bases by first derivatizing the solid support base with a linker. Such linkers are well known to one of ordinary skill in the art and include compounds of the formulas Qxe2x80x94Rxe2x80x94SH and QRSi(OR)xRyXz, where
Q comprises a moiety of the formula: xe2x80x94SH, xe2x80x94CH(O)CH2, xe2x80x94NR2, xe2x80x94NRxe2x80x2xe2x80x94(CH2)nxe2x80x94NR2;
each of Rxe2x80x2 and R is independently hydrogen or an alkyl group;
X is a halide; and
each of x, y, and z is independently an integer from 0 to 3, provided the sum of x, y, and z is 3.
Particular exemplary linkers include aminoalkyltrialkoxysilanes, and those disclosed below.